scholarly journals Expression of I2PP2A, an inhibitor of protein phosphatase 2A, induces c-Jun and AP-1 activity

1999 ◽  
Vol 341 (2) ◽  
pp. 293-298 ◽  
Author(s):  
S W.K. AL-MURRANI ◽  
James R. WOODGETT ◽  
Zahi DAMUNI
Keyword(s):  
Dna Binding ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Dependent Manner ◽  
Activator Protein 1 ◽  
Intact Cells ◽  
Hek 293 ◽  
293 Cells ◽  
Site Selectivity ◽  
Phosphatase 2A

Transient expression of I2PP2A, a potent inhibitor of protein phosphatase 2A (PP2A), in HEK-293 cells increased the concentration and DNA binding of the proto-oncogene c-Jun. In contrast, expression of the catalytic subunit of PP2A (PP2AC) markedly decreased the concentration and DNA binding of c-Jun. Expression of I2PP2A also increased the transcriptional activity of activator protein-1, and this effect was diminished in a dose-dependent manner by expression of PP2AC. Densitometric analysis following Western blotting of extracts with antibodies specific for phospho-Ser63 and Ser73 suggests that the effects of I2PP2A and PP2AC expression might be mediated, in part, by changes in the phosphorylation of c-Jun at Ser63. The results indicate that I2PP2A elicits effects that are consistent with it acting as an inhibitor of PP2A in intact cells, and suggest that PP2A might exhibit site selectivity with respect to c-Jun phosphorylation.

scholarly journals Protein phosphatase 2A regulates life and death decisions via Akt in a context-dependent manner

2007 ◽  
Vol 104 (48) ◽  
pp. 19011-19016 ◽  
Author(s):  
S. Andrabi ◽  
O. V. Gjoerup ◽  
J. A. Kean ◽  
T. M. Roberts ◽  
B. Schaffhausen
Keyword(s):  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Dependent Manner ◽  
Life And Death ◽  
Phosphatase 2A ◽  
Context Dependent

Regulation by glucose and calcium of the carboxylmethylation of the catalytic subunit of protein phosphatase 2A in insulin-secreting INS-1 cells

2004 ◽  
Vol 286 (6) ◽  
pp. E1032-E1041 ◽  
Author(s):  
Rengasamy Palanivel ◽  
Rajakrishnan Veluthakal ◽  
Anjaneyulu Kowluru
Keyword(s):  
Insulin Secretion ◽  
Glucose Metabolism ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Catalytic Subunit ◽  
Inhibitory Effects ◽  
Β Cells ◽  
Intact Cells ◽  
Phosphatase 2A ◽  
Glucose Metabolites

Previously, we reported that the catalytic subunit of protein phosphatase 2A (PP2Ac) undergoes carboxylmethylation (CML) at its COOH-terminal leucine, and that inhibitors of such a posttranslational modification markedly attenuate nutrient-induced insulin secretion from isolated β-cells. More recent studies have suggested direct inhibitory effects of glucose metabolites on PP2A activity in isolated β-cells, implying that inhibition of PP2A leads to stimulation of insulin secretion. Because the CML of PP2Ac has been shown to facilitate the holoenzyme assembly and subsequent functional activation of PP2A, we investigated putative regulation by glucose of the CML of PP2Ac in insulin-secreting (INS)-1 cells. Our data indicated a marked inhibition by specific intermediates of glucose metabolism (e.g., citrate and phospho enolpyruvate) of the CML of PP2Ac in INS-1 cell lysates. Such inhibitory effects were also demonstrable in intact cells by glucose. Mannoheptulose, an inhibitor of glucose metabolism, completely prevented inhibitory effects of glucose on the CML of PP2Ac. Moreover, glucose-mediated inhibition of the CML of PP2Ac was resistant to diazoxide, suggesting that glucose metabolism and the generation of glucose metabolites might control inhibition of the CML of PP2Ac. A membrane-depolarizing concentration of KCl also induced inhibition of the CML of PP2Ac in intact INS cells. On the basis of these data, we propose that glucose metabolism and increase in intracellular calcium facilitate inhibition of the CML of PP2Ac, resulting in functional inactivation of PP2A. This, in turn, might retain the key signaling proteins of the insulin exocytotic cascade in their phosphorylated state, leading to stimulated insulin secretion.

Propofol Inhibits Phosphorylation of N -methyl-d-aspartate Receptor NR1 Subunits in Neurons

2006 ◽  
Vol 104 (4) ◽  
pp. 763-769 ◽  
Author(s):  
Seth Kingston ◽  
Limin Mao ◽  
Lu Yang ◽  
Anish Arora ◽  
Eugene E. Fibuch ◽  
...  
Keyword(s):  
Glutamate Receptors ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Cortical Neurons ◽  
Protein Phosphatase 2A ◽  
Ionotropic Glutamate Receptors ◽  
Dependent Manner ◽  
Calyculin A ◽  
General Anesthetic ◽  
Phosphatase 2A

Background Anesthetics may interact with ionotropic glutamate receptors to produce some of their biologic actions. Cellular studies reveal that the ionotropic glutamate receptors, N-methyl-D-aspartate receptors (NMDARs), can be phosphorylated on their NR1 subunits at the C-terminal serine residues, which is a major mechanism for the regulation of NMDAR functions. It is currently unknown whether anesthetics have any modulatory effects on NMDAR NR1 subunit phosphorylation. Methods The possible effect of a general anesthetic propofol on phosphorylation of NR1 subunits at serine 897 (pNR1S897) and 896 (pNR1S896) was detected in cultured rat cortical neurons. Results Propofol consistently reduced basal levels of pNR1S897 and pNR1S896 in a concentration-dependent manner. This reduction was rapid as the reliable reduction of pNR1S896 developed 1 min after propofol administration. Pretreatment of cultures with the protein phosphatase 2A inhibitors okadaic acid or calyculin A blocked the effect of propofol on the NR1 phosphorylation, whereas okadaic acid or calyculin A alone did not alter basal pNR1S897 and pNR1S896 levels. In addition, propofol decreased tyrosine phosphorylation of protein phosphatase 2A at tyrosine 307, resulting in an increase in protein phosphatase 2A activity. In the presence of propofol, the NMDAR agonist-induced intracellular Ca2+ increase was impaired in neurons with dephosphorylated NR1 subunits. Conclusions Together, these data indicate an inhibitory effect of a general anesthetic propofol on NMDAR NR1 subunit phosphorylation in neurons. This inhibition was mediated through a signaling mechanism involving activation of protein phosphatase 2A.

Protein Phosphatase 2A (PP2A) Associates with Integrin αIIbβ3 and Regulates Adhesion to Fibrinogen.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1644-1644
Author(s):  
Kadekuzhi V. Vijayan ◽  
Yan Liu ◽  
Paul F. Bray
Keyword(s):  
Protein Phosphatase ◽  
Platelet Adhesion ◽  
Protein Phosphatase 2A ◽  
Critical Role ◽  
Specific Interaction ◽  
Cytoskeletal Proteins ◽  
Dependent Manner ◽  
Integrin Αiibβ3 ◽  
Knock Down ◽  
Phosphatase 2A

Abstract We have previously demonstrated that protein phosphatase 1 (PP1) associates constitutively with the integrin αIIb subunit and regulates myosin light chain phosphorylation. In this study, we considered whether other member of the serine/threonine (Ser/Thr) phosphatase family namely, protein phosphatase 2A (PP2A) associates with the integrin αIIbβ3. Co-immunoprecipitation assays using lysates from resting human platelets revealed the presence of a catalytic subunit of PP2A (PP2Ac) in the αIIb immunoprecipitates, and in a reciprocal experiment, αIIb was detected in the PP2Ac immunoprecipitates. In contrast, another platelet abundant Ser/Thr phosphatase, protein phosphatase 2C (PP2C) was not detected in the αIIb immunoprecipitates. Furthermore, the association of PP2Ac with integrin αIIbβ3 was also observed in 293 cells overexpressing αIIbβ3. These results indicate a constitutive and specific interaction of PP2Ac with the integrin αIIbβ3. Polystyrene beads coated with purified PP2Ac but not BSA supported the binding of purified integrin αIIbβ3 in a dose dependent manner, suggesting that the interaction of PP2Ac with αIIbβ3 was direct. Furthermore, purified PP2Ac as well as PP2Ac in lysates obtained from the resting platelets bound specifically to a biotinylated αIIb cytoplasmic peptide encompassing residues 985–995 but not to a scrambled peptide, suggesting that the integrin αIIb is sufficient to mediate the interaction of PP2Ac in vitro. The association of PP2Ac with the platelet integrin αIIbβ3 was not altered during platelet adhesion to fibrinogen (αIIbβ3 outside-in signaling) or during thrombin or ADP stimulation (inside-out signaling to αIIbβ3). In contrast, we have previously shown that integrin-bound PP1 dissociated from the αIIbβ3 complex upon platelet adhesion and thrombin-induced platelet activation. The association of PP2Ac with the integrin αIIbβ3 correlates well with the dephosphorylation of a PP2A substrate, extracellular-signal regulated kinase 2 (ERK2) during thrombin-induced platelet aggregation that we and others have previously demonstrated. More importantly, ERK2 dephosphorylation was not observed in platelets from Glanzmann thrombasthenic patients or in normal platelets pretreated with RGDS or integrilin, suggesting a critical role for integrin αIIbβ3 in the dephosphorylation of ERK2. To ascertain a physiological relevance for the PP2A-αIIbβ3 association, we used short interfering RNAs (siRNAs) to knock down the expression of PP2Ac in the 293/αIIbβ3 cells. Knock down was maximal (~55–70%) and specific to PP2Ac because PP1 and actin protein levels were not different between the control and PP2Ac siRNA treated cells. Consistent with the reduction in the PP2Ac protein level in the PP2Ac knock down cells there was ~70% reduction in the PP2Ac phosphatase activity, and a concomitant increased basal ERK2 phosphorylation. PP2Ac knock down significantly (P≤0.006) increased the adhesion of 293/αIIbβ3 cells to fibrinogen. The adhesion was αIIbβ3 specific because it could be abolished with an αIIbβ3 function blocking antibody (10E5). These findings supports a mechanism whereby the integrin associated Ser/Thr phosphatases might regulate αIIbβ3 adhesive functions via dephosphorylation of key cytoskeletal proteins.

Cross Talk Between Serine/Threonine Protein Phosphatase 2A and Tyrosine Kinase Src Regulates αIIbβ3 Function

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2871-2871
Author(s):  
Subhashree Pradhan ◽  
Vimal Patel ◽  
K. Vinod Vijayan
Keyword(s):  
Tyrosine Kinase ◽  
Cross Talk ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Tyrosine Phosphatase ◽  
Catalytic Subunit ◽  
Cellular Functions ◽  
Small Interference Rna ◽  
293 Cells ◽  
Phosphatase 2A

Abstract Protein phosphatase 2A (PP2A) is a ubiquitously expressed serine/threonine phosphatase that regulates a variety of cellular functions. In the context of the platelets, we have previously shown that a pool of the catalytic subunit of PP2A (PP2Ac) associates constitutively with the resting αIIbβ3 and negatively regulates αIIbβ3 signaling. However, the mechanism by which PP2Ac controls αIIbβ3 adhesive function is incompletely understood. In this study, we demonstrated that PP2Ac expressed as a GST fusion protein interacts with the tyrosine kinase Src. Activation of Src is essential to initiate αIIbβ3 outside-in signaling. Small interference RNA mediated knockdown of endogenous PP2Acα expression in 293 cells overexpressing αIIbβ3 (293-αIIbβ3) and murine megakaryocytes, resulted in the activation of Src, as evidenced by the dephosphorylation of Src Tyr-529 and phosphorylation of Src Tyr-418. In contrast to PP2Acα, knockdown of the catalytic subunit of protein phosphatase 1 (PP1cα) did not activate Src, indicating that the regulation of Src activity by PP2Ac is specific. Dephosphorylation of Src Tyr-529 was not observed in PP2Aca depleted 293 cells treated with sulfanamido-benzbromarone compound, a selective protein tyrosine phosphatase 1 (PTP-1) inhibitor. These results suggest that inhibition of PP2Ac may activate a tyrosine phosphatase, capable of dephosphorylating Src Tyr-529. Activation of Src in PP2Ac depleted 293-αIIbβ3 cells had functional consequences for integrin αIIbβ3. PP2Ac depleted 293-αIIbβ3 cells exhibited ~2 fold increased adhesion to immobilized fibrinogen. Inhibition of Src kinase with a pharmacological agent PP2 but not by PP3 an inactive analogue of PP2, abolished the increased adhesiveness of PP2Acα–depleted 293 cells to fibrinogen. Finally, the increased activation of extracellular signal-regulated kinase (ERK1/2) in PP2Acα-depleted cells that we previously demonstrated was also blocked by Src inhibitor PP2 but not by PP3. These results indicate that both Src and ERK1/2 are activated in response to PP2Ac inhibition, with activation of Src being upstream of ERK1/2. These studies illustrate that inhibition of PP2Ac promotes αIIbβ3 adhesiveness by activating Src, and imply that the control of αIIbβ3 adhesive function can be further fine-tuned by a cross talk between the serine/threonine phosphatase PP2A and the tyrosine kinase Src.

scholarly journals Regulation of Cancerous inhibitor of PP2A (CIP2A) by small molecule inhibitor for c-Jun NH2-Terminal Kinases (JNKs), SP600125, in Human Fibrosarcoma (HT1080) cells

F1000Research ◽  
2013 ◽  
Vol 2 ◽  
pp. 174
Author(s):  
Anchit Khanna
Keyword(s):  
Protein Expression ◽  
Small Molecule ◽  
Protein Phosphatase ◽  
Affinity Purification ◽  
Protein Phosphatase 2A ◽  
Small Molecule Inhibitor ◽  
Dependent Manner ◽  
Molecule Inhibitor ◽  
Ht1080 Cells ◽  
Phosphatase 2A

Background: Protein phosphatase 2A inhibition is one of the pre-requisites for human cell transformation. Previously, we have identified an endogenous inhibitor of PP2A, CIP2A (Cancerous Inhibitor of Protein Phosphatase 2A) in human fibrosarcoma cells (HT1080) using tandem affinity purification. CIP2A over expression has been demonstrated in almost every tumour type studied so far. However, our understanding on the mechanisms regulating CIP2A expression in human cancers, especially in sarcomas, is still emerging. Methods:  Human fibrosarcoma (HT1080) cells were treated with small molecule inhibitors against the three major signalling pathways, namely p38, MEK and JNK pathways to identify the pathway regulating CIP2A expression in the sarcoma cells. This was followed by verification of the results using small interfering RNAs (siRNA) for the kinases. Results:  In line with previous observations, small molecule inhibitor for MEK pathway (PD98059) decreased CIP2A mRNA and protein expression. Interestingly, small molecule inhibitor for the JNK pathway, SP600125 decreased mRNA and protein levels of CIP2A oncoprotein with negligible effect of SB203580 (p38 kinase) inhibitor on CIP2A expression in HT1080 cells. However, siRNAs specific to either JNK1 or JNK2 kinases did not result in decrease in CIP2A expression. Contrarily, two different CIP2A siRNAs, which were used as positive controls, decreased JNK2 expression in HT1080 cells. Conclusion: Although it is well established that SP600125 inhibits JNK kinases, it has also been shown to inhibit a spectra of other kinases. SP600125 inhibits CIP2A protein expression both in time and concentration dependent manner. However, depletion of both JNK1 and JNK2 kinases using specific siRNAs fails to decrease CIP2A protein expression levels, thereby indicating the need to verify the results obtained by treatment with small molecular inhibitors of kinases by independent approaches like two different target specific siRNAs. Finally, fortuitously we identify JNK2 as a CIP2A downstream target in HT1080 cells.

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